Bar = 20 μm. J. Biol Chem 277, 43888–43894 (2002). Coexpressed RFP was not detected in the purified chloroplast fraction, confirming that it was not contaminated by cytoplasmic proteins. Bar = 20 μm. These results raised the possibility that T4A/T7A is trapped in the import channel, although we cannot completely exclude the possibility that it may form a high Mr complex. 4C, e). In addition, the double-T deletion mutants, ΔT2/T3, ΔT3/T4, ΔT3/T6, ΔT4/T7, and ΔT6/T7 were generated (Fig. Identification of critical sequence motifs in T3, T6, and T7. However, we cannot rule out the possibility that these regions are involved in binding to the lipid membranes of chloroplasts (Pilon et al., 1995; Pinnaduwage and Bruce, 1996), as well as to the guidance complex (May and Soll, 2000). Leaf tissues were harvested from 2-week-old plants and used immediately for protoplast isolation. To investigate the sequence motifs in the cTPs, Ala substitution mutants of the Arabidopsis (Arabidopsis thaliana) RbcS transit peptide were generated. Ala substitution or deletion of T4 causes targeting efficiency to drop to 40% to 50% of the wild-type level. B, Localization of reporter proteins. In addition, Ala substitution of both T2 and T7 causes 40% to 50% of the total reporter protein to remain in the intermediate form, almost identical to the result with T4A alone. These proteins are synthesized in the cytoplasm as precursors containing an N-terminal chloroplast targeting signal, the transit peptide, that mediates post-translational import into the chloroplast. C, Protein extracts from protoplasts transformed with T4A/T7A:GFP were treated with 1.0% Triton X-100 treatment on ice and separated into soluble (S) and membrane (P) fractions by ultracentrifugation. Arrows indicate the GFP signals in the cytoplasm. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.074575. T4A:GFP showed a western-blot pattern that differed from the other mutants, as well as from the wild type. Various chloroplast transit peptides (CTP) have been used to successfully target some foreign proteins into chloroplasts, but for other proteins these same CTPs have reduced localization efficiencies or fail completely. These results indicated that the T7 motif was involved in a later step than the T6 motif and may play a role in crossing the envelope membrane. The data herein clearly demonstrate that the transit peptides have multiple sequence elements with complex relationships. A target peptide is a short (3-70 amino acids long) peptide chain that directs the transport of a protein to a specific region in the cell, including the nucleus, mitochondria, endoplasmic reticulum (ER), chloroplast, apoplast, peroxisome and plasma membrane.. The intensities of protein bands were quantified using image capture software. Similar to T3A/T7A:GFP, T4A/T7A:GFP produced no GFP signal (data not shown). Figure 5. In vivo targeting and western-blot analysis of double-T block Ala substitution mutations in protoplasts. These results strongly suggested that in the absence of the T4 motif, the reporter protein had difficulty passing through the import channel. 1C), indicating that these mutant transit peptides were able to deliver reporter proteins into chloroplasts. 2020 Aug 27;8:e9772. To examine membrane association of the T4A:GFP intermediate, protein extracts were treated with Na2CO3 or Triton X-100 and fractionated by ultracentrifugation. 7C). Protein import experiments revealed that each protein contained transit peptide–specific sequence motifs critical for protein import into chloroplasts. The underlying cause of the failures remains an open question, and more effective CTPs are needed. The processing site is located in T6 (Gavel and von Heijne, 1990) and mutations at or near that site may cause processing to occur at alternative sites. The nature of this putative complex was unclear. Thus, it has been suggested that the cTP is composed of multiple domains that may have different roles and/or overlapping functions that are required for import across the two chloroplast membranes (von Heijne et al., 1989; Pilon et al., 1995; Rensink et al., 1998, 2000). Under the same conditions, aleurain and Toc75 were detected in the supernatant and pellet fractions, respectively, confirming that the fractionation experiment had been performed properly. Bar = 20 μm. A, Sequences of double-T mutations with partially restored amino acid sequences. B, Localization of reporter proteins. In certain cases, RFP was cotransformed into protoplasts together with GFP constructs. Plastid transit peptides-where do they come from and where do they all belong? Generation of an improved rCTP and its performance in transgenic rice plants. This site needs JavaScript to work properly. The GFP patterns of T2A/T5A:GFP, T2A/T6A:GFP, and T2A/T8A:GFP were nearly identical to that of the wild type (data not shown). Western-blot analysis confirmed this observation: T4A/T7A:GFP was present as the 37-kD intermediate at 12 h AT (Fig. Figure 3. 7C, d) as observed with T4A/T7A:GFP (Fig. Western-blot analysis showed that approximately 80% of the T3A/T7A:GFP was in the intermediate form at 12 h AT, with the remaining protein in M (Fig. These results were quite similar to those obtained with the individual-T Ala substitution mutants. Protein extracts from protoplasts transformed with the indicated constructs at the indicated time points were used for western-blot analysis. In vivo targeting of deletion mutants in protoplasts. As a control for chloroplast fractionation, RFP (red fluorescent protein) was cotransformed into the protoplasts together with T4A + RK:GFP. 4C, j). CAS In addition, as indicated above, FP is part of the sequence that is similar to the proposed binding site of 14-3-3 (May and Soll, 2000; Bruce, 2001).The western-blot patterns obtained with T4A + RK:GFP and T4A + FP:GFP differ slightly: T4A + FP:GFP yields only M, whereas T4A + RK:GFP yields a small amount of 36- and 37-kD intermediates together with a very slowly migrating protein species of 65 kD, in addition to M. These protein species are tightly associated with the particulate fraction and are resistant to solubilization by Triton X-100 or Na2CO3. Protein extracts from protoplasts at 6 to 36 h AT were analyzed by western blotting. 1C, e). Sequence analysis has revealed that 75% of 727 plastid precursor proteins contain at least one DnaK-binding site (Rial et al., 2000). 2020 Sep;18(9):1882-1896. doi: 10.1111/pbi.13347. 7A). These constructs were introduced into a pUC-based expression vector. ΔT3/T4:GFP and ΔT3/T6:GFP were detected primarily in the supernatant fraction, whereas ΔT4/T7:GFP and ΔT6/T7:GFP yielded significant amounts of reporter protein in the pellet (Fig. In the presence of thermolysin, the 37-kD form was converted to a 31-kD form, which may have been due to the resistance of the GFP domain to thermolysin (Fig. 2, B, c–f, C, b and c) when examined by image and western-blot analyses, indicating that D1 and D2 of T1 were functionally redundant. T3A/T6A:GFP produced diffuse GFP signals in the cytoplasm, but not in the chloroplasts, at both 12 and 24 h AT (Fig. The results shown are representative of at least three independent western-blot analyses. In vitro and in vivo analyses have shown that DnaK/HSP70 binds to its predicted binding sites in the cTP of the small subunit of the Rubisco complex (RbcS) and the ferredoxin-NADP+ reductase precursor (Ivey and Bruce, 2000; Ivey et al., 2000; Rial et al., 2000). To further examine the importance of these hydrophobic residues in the targeting, we generated two additional mutants, T1ML/AA and T1MLM/AAA, which had Ala substitutions of two and three hydrophobic residues in T1 in the wild-type background, respectively (Fig. As expected, aleurain was detected in the supernatant, whereas γ-COP and AtVSR were detected in the pellet fraction. 8; Williamson and Slocum, 1994). 2A). When protein extracts were fractionated, Pr was equally distributed in the supernatant and pellet fractions (Fig. Using cross-validation, 88% of the sequences in our homology reduced training set were correctly classified as transit peptides or nontransit peptides. This interaction may be important for initial binding of the preprotein to chloroplasts, which subsequently leads to diffusion of the preprotein to the receptor complex (van't Hof and de Kruijff, 1995; Chen and Li, 1998). 5B, d), indicating that chloroplast association was defective, but not to the extent observed with T3A/T6A:GFP. Int J Mol Sci. Immunohistochemistry with the anti-GFP antibody was used to localize T3A/T7A:GFP. The 37-kD form of T4A/T7A:GFP was resistant to solubilization by Triton X-100 (Fig. Pr, Precursor; M, mature form; and asterisk (*), unknown 65-kD form. The 3′ fragments were amplified using the upper primers and the common nos-terminator primer, and thus contained the 18 bps 5′ to the mutated region, the mutated region, the GFP coding region, and the nos terminator. Analyses showed that RC2 can more effectively target diverse proteins, including EcTSR and EcGCL, into rice chloroplasts. The other combinations of double-T mutants were examined for their ability to target the reporter protein to chloroplasts. Under the conditions used in these experiments, only Ala substitution of T4 significantly affects the targeting efficiency. At 12 AT, 40% to 50% of the total reporter protein accumulated as the 37-kD form. In agreement with the image analysis, the intermediate form was detected primarily in the pellet (Fig. Epub 2019 May 22. 5B, a and b). These data indicated that a large number of Ser residues in T1 were detrimental for targeting. In addition, the KK motif in the transit peptide of ATCase may be equivalent to the RK motif in the transit peptide of RbcS. HSP70 or 14-3-3 may bind to motifs in the transit peptide, as proposed previously (Ivey et al., 2000; May and Soll, 2000; Rial et al., 2000). Protein extracts from protoplasts transformed with the indicated constructs at the indicated time points were used for western-blot analysis. -, Richter S. & Lamppa G. K. Determinants for removal and degradation of transit peptides of chloroplast precursor proteins. These results suggested that T3A/T8A:GFP accumulated at the chloroplast envelope membrane at early time points and was imported into chloroplasts over time, at a slower rate than the wild-type protein. Green, red, and yellow signals indicate GFP, autofluorescence of chlorophyll, and the overlap between green and red fluorescent signals, respectively. However, the sequence information encoded by transit peptides is not fully understood. A transit peptide-like sorting signal at the C terminus directs the Bienertia sinuspersici preprotein receptor Toc159 to the chloroplast outer membrane. Protein extracts were fractionated and detected as described for Figure 1D. In addition, protein extracts were fractionated and detected as described for Figure 1D. Both mutant transit peptides translocated the reporter proteins into chloroplasts as efficiently as the wild type (Fig. & Herrmann R. G. Domain structure of mitochondrial and chloroplast targeting peptides. The single-T deletion mutants, with the exception of ΔT4, primarily yielded the M (Fig. The 36-, 37-, and 65-kD forms, but not the 31-kD form, were sensitive to thermolysin (Fig. In a previous study, the LKSS motif in the transit peptide of ferredoxin-2 was shown to be part of the binding site of a 14-3-3 protein (Pilon et al., 1995). In addition, the intermediate form of ΔT4/T7:GFP was also sensitive to a protease (Fig. In the case of ΔT4, the reporter proteins displayed a complex pattern with four protein species at 31, 32, 34, and 35 kD (Fig. HHS Both the 37-kD form and the M were detected in the pellet but not in the supernatant (Fig. 7B, c). Green, red, and yellow signals indicate GFP, autofluorescence of chlorophyll, and the overlap between green and red fluorescent signals, respectively. 3D, a). Classical examples and original systems (referenced) are shown. Considerable effort has been devoted to elucidating the molecular mechanisms of protein import into various parts of chloroplasts (Keegstra and Froehlich, 1999; Bauer et al., 2000; Jarvis and Robinson, 2004; Kessler and Schnell, 2004; Soll and Schleiff, 2004; Hofmann and Theg, 2005). Epub 2018 Feb 27. These constructs were transiently expressed in protoplasts to examine their targeting to chloroplasts. 7C), as observed with double-T mutants with Ala substitutions in the corresponding regions. 4C, a and b), indicating that motifs in T3 and T6 were important for the binding to chloroplasts. Furthermore, as expected, membrane-associated γ-COP was completely solubilized by 0.1 m Na2CO3 and the integral membrane protein AtVSR1 could only be completely solubilized by 1.0% Triton X-100. Nuclear-encoded plastid proteins can be divided into two groups depending on whether or not they possess a cleavable transit peptide. Transit peptide sequences located in the N-terminus of these proteins facilitate the transfer from the cytoplasm where they are synthesized, back to the chloroplast organelle (1 ; reviewed in 2). Next, we examined accessibility of T4A/T7A:GFP to thermolysin and found that the 37-kD form of T4A/T7A:GFP was thermolysin sensitive (Fig. Pr, Precursor form; M, mature forms; and h, hour. These results demonstrate that the transit peptide contains multiple motifs and that some of them act in concert or synergistically. 4C, k). Get the latest public health information from CDC: https://www.coronavirus.gov, Get the latest research information from NIH: https://www.nih.gov/coronavirus, Find NCBI SARS-CoV-2 literature, sequence, and clinical content: https://www.ncbi.nlm.nih.gov/sars-cov-2/. However, when T2A is combined with T4A, the reporter proteins accumulate at the envelope membrane as intermediate forms, indicating that T2 and T4 act together to translocate a protein into chloroplasts. In agreement with data from image analysis, T3A/T6A:GFP primarily yielded the Pr with only a small portion of M at 12 h AT (Fig. At 24 h AT, the GFP signals were present in the chloroplasts (Fig. Zhang Z, Liang X, Lu L, Xu Z, Huang J, He H, Peng X. BMC Plant Biol. To further characterize the distribution of T2A/T4A:GFP and T2A/T7A:GFP, protein extracts were separated into supernatant and pellet fractions by ultracentrifugation. 1B, d). Thus, one possibility is that the mutant transit peptide T3A/T7A can bind to the initial binding component of the receptor complex via the intact T6 and may engage in translocation, but cannot cross the envelope membrane efficiently due to the absence of the T7 motif. USA.gov. 2C, j and k). In this study, we initially observed that two E.coli enzymes, EcTSR and EcGCL, failed to be targeted into rice chloroplasts by the commonly-used rice rbcS transit peptide (rCTP) and were subsequently degraded. 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